Meat quality in broiler chickens fed on cowpea (Vigna unguiculata [L.] Walp) seeds

The study aimed to evaluate the effects of a diet containing untreated cowpea (CWP; Aura 26 variety) seeds as a protein source on quality parameters of chickens’ breast (PM; Pectoralis major) and thigh muscles (BF; Biceps femoris). A total of 240 Ross 308 broiler chickens were randomly allotted to two groups: a control group fed with soybean meal (SBM) and an experimental group fed with CWP included at 200 g/kg as a replacement of SBM. Each group consisted of six pens as replicates, with 20 chicks per pen. At 6 weeks of age, twelve birds/group were slaughtered. Compared to SBM group, the group fed CWP had higher (P < 0.0001) lightness (L*) and redness (a*) values of PM and BF muscles, the latter had also higher yellowness (b*, P < 0.0001). The collagen and protein contents were significantly higher in CWP group in both PM and BF muscles, while fat was lower (P < 0.001) only in BF muscle. The use of CWP into broilers’ diets did not negatively impact the textural properties, i.e., hardness, adhesiveness, cohesiveness, springiness, gumminess, chewiness, and resilience of PM and BF, showing similar values in both groups. Also, PM and BF muscles of birds fed CWP had significantly higher (P < 0.05) levels of C:18:3n-3 and C:20:5n-3 compared with birds fed SBM. The n-6/n-3 PUFA ratio was significantly lower in CWP group (11.72 and 7.00) compared to SBM (13.47 and 12.63) for both PM and BF muscles. These results indicate that CWP can be considered a promising protein source for broiler chickens’ feed.

www.nature.com/scientificreports/ The study aimed to compare the meat traits, including physicochemical and textural properties as well as fatty acids composition, in broiler chickens fed on diets containing untreated CWP seeds as an alternative to SBM.

Results
There were no significant differences (P > 0.05) in the values of pH 24 for the breast muscles (PM; Pectoralis major) obtained from birds in the experimental group, which was fed with CWP compared to the control group (SBM ;  Table 1). However, the values of colour parameters for PM, such as lightness (L*) and redness (a*) from birds fed CWP, were significantly different (P < 0.0001) than in the SBM group, while the yellowness (b*) tended the increase (P = 0.070). Specifically, muscles from CWP group were characterized by higher L*, a* and b* (CWP: 62.47, 13.35 and 13.61; SBM: 55.44, 10.87 and 12.27, respectively). The analysis of the chemical composition of PM also showed significant differences in the content of protein (P < 0.0001) as well as collagen (P < 0.0001). No differences (P > 0.05; Table 1) were found in PM's moisture, and the fat level of broilers fed the SBM and CWP diets.
The thigh muscles (BF; Biceps femoris) obtained from birds fed with CWP were characterized by comparable pH 24 after slaughter than the SBM group (Table 1). All color values of BF from birds fed CWP were significantly higher (P < 0.0001) compared to birds fed SBM. However, the lightness (L*) redness (a*) and the yellowness (b*) of BF differed depending on the source of protein in the feed. Moreover, dietary protein sources influenced the BF protein, fat, and collagen content (Table 1). BF muscles collected from broilers fed CWP diet registered a significantly higher (P = 0.016) content of protein as well as collagen (P < 0.0001), while the fat level was lower (P < 0.001) compared with birds fed SBM diet. There were no significant differences (P > 0.05) in the moisture content for the BF muscles between broilers fed SBM and CWP diets.
The effect of different protein sources on the texture profile analysis (TPA) of PM and BF is shown in Table 2. Overall, the dietary inclusion of CWP into broilers' diets did not negatively impact the TPA attributes of PM and BF, which showed similar values in both groups. Hardness, adhesiveness, cohesiveness, springiness, gumminess, chewiness, and resilience values were not different (P > 0.05) among groups in PM and BF samples. Data in Tables 3 and 4 indicate that the use of different protein sources in the diet of broiler chickens can influence the FA composition in PM and BF samples. As expected, the predominant FA in the PM and BF of the broiler chickens fed both the SBM and CWP diets was C18:1n9 (PM: 34.89 vs 35.14% of total FA, respectively; BF: 38.87 vs 37.72%, respectively), followed by C16:0 (PM: 21.21 vs 22.30% of total FA, respectively; BF: 27.00 vs 25.12%, respectively) and C18:2n-6 (PM: 22.74 vs 22.38% total FA; BF: 15.07 vs 15.82% total FA). Alphalinolenic acid (ALA, C18:3n-3) is the precursor of long-chain n-3 PUFA such as eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (DPA, C22:5n-3), and docosahexaenoic acid (DHA, C22:6n-3), which are commonly referred to as n-3 or omega-3 PUFA. In this study, PM from birds fed CWP diets were characterized by a significantly higher content of ALA (P = 0.005) and EPA (P = 0.016) compared to the SBM diet (Table 3). Moreover, the proportion of total n-3 PUFA was slightly higher in chickens fed CWP (2.20) than in chickens fed SBM (2.00), but the difference was not significant (P > 0.05). The n-6/n-3 PUFA ratio was significantly lower (P = 0.045) in breast muscles from CWP group (11.72) compared to SBM (13.47). There were no significant www.nature.com/scientificreports/ differences (P > 0.05) in the total SFA and MUFA contents for the PM muscles between broilers fed SBM and CWP diets, although feeding CWP resulted in a significantly higher (P < 0.01) content of C14:0 and C16:0, while the C18:0 was lower (P = 0.002; Table 3). Likewise, the use of different protein sources in the diet of broiler chickens also, influenced the FA compositions in BF samples ( Table 4). The significantly higher (P = 0.018) content of ALA and C18:4n-3 (P < 0.0001) as well as EPA (P = 0.006) and total n-3 PUFA content (P = 0.011) was observed in chickens' BF muscle fed CWP compared with the SBM diet. The analysis of BF samples also, revealed a significantly lower (P = 0.002) n-6 to n-3 PUFA ratio in broilers fed CWP diet (1.30) compared to SBM (7.00). Regarding the content of total SFA and MUFA in BF muscles, they followed the same trend as those in PM samples (P > 0.05).

Discussion
For both groups, PM and BF, pH 24 values were in the range of raw meat (5.5-6.2) and similar to other studies 14 who reported close values for breast muscle. This indicates that there were no quality problems with the breast meat from each dietary treatment group.
Meat colour is an essential quality parameter since it is directly perceived by the consumer. In our study, the change of protein source in the diet significantly influenced the colour of breast and thigh muscle. This has been confirmed by Laudadio et al. 15 , who reported differences in the values of redness (a*) and the yellowness (b*) of breast and leg muscles, which were higher in chickens fed a diet with the inclusion of other legume plants (i.e., faba bean) compared to broiler fed SBM. The authors suggested that higher values of yellowness (b*) in leg muscles could be attributed to different FA contents and the SFA/ PUFA ratio in these muscles. Another studies 16,17 on pea or lupin as a substitute for SBM into broilers' diets also reported that the yellowness (b*) of leg muscles was significantly higher, suggesting a lower level of lipids. Likewise, a trend towards higher lightness (L*) of leg muscles was also reported by Kuzniacka et al. 18 , who investigated the effect of faba bean (Vicia faba var. minor) based diet. Our study found significantly higher values of lightness (L*), redness (a*) and yellowness (b*) for PM and BF from chickens fed a diet based on CWP. Even if BF of CWP group showed a higher lightness, the pH value was in the range for normal meat.
In our study, the use of CWP as a substitute for SBM had a significant effect on most of the chemical parameters (the content of protein, fat and collagen) of breast and thigh muscles from 6-weeks-old broiler chickens. Muscles collected from broilers fed CWP diet registered higher protein and collagen contents. Consistently with our study, Laudadio et al. 15 reported significantly higher content of collagen in breast and leg muscles from broilers fed a diet containing dehulled-micronized fava beans (310 g/kg). On the other hand, Dal Bosco et al. 19 reported significantly lower fat content in leg muscles from organic slow-growing chickens fed a diet containing faba bean, which was also confirmed in our study. There is a lack of relative information regarding the texture properties of CWP, and indeed, of legume plants. To the best of our knowledge, this is the first study about the influence of the CWP diet on texture attributes of muscles. Therefore, more study is needed to determine the effects of these seeds on textural properties in broiler meat. The TPA breast meat traits observed in the present research were overall satisfactory and in line with values reported for another legume, i.e., chickpea 20 . www.nature.com/scientificreports/ Manipulating broiler chickens' diets by adding different protein sources resulted in significant changes in the FA composition of breast and thigh muscle. No research appears to have been reported on the effect of raw CWP seeds on meat FA composition in broiler chickens. The present study provides new insights into the use of CWP in the diet of broiler chickens. Therefore, this subject should be considered a new investigation. The use of CWP influenced markedly the breast and thigh muscle FA content. The highest total n-3 PUFA content was observed in the chickens' breast (2.20 vs 2.00) and thigh muscle (2.58 vs 1.34) fed CWP compared to SBM. This result was associated with a significant increase in most of the n-3 FA's in PM and BF samples, especially for those that may have potential benefits to human nutrition, i.e., ALA and EPA. Similar results were noted in other experiments where broiler chickens were fed diets with different protein sources such as faba bean 15 and raw lentil seeds 21 as replacements for SBM. The authors of the cited studies reported higher total content of n-3 PUFA in the breast Table 3. Fatty acids composition (% of total fatty acids) in breast muscles from 6-week-old broiler chickens. SEM standard error of the mean. A Group SBM: feed based on soybean meal; group CWP: feed based on raw cowpea seeds (Vigna unguiculata [L.] Walp, Aura 26 cv.). B Total SFA: saturated fatty acids, sum of C8:0 + C10:0 + C12:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0 + C24:0. C Total MUFA: monounsaturated fatty acids, sum of C14:1 + C15:1 + C16:1 + C17:1 + C18: 1n-9 + C22:1n-9 + C24: 1n-9. D Total n-6 PUFA: sum of C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:2n-6 + C22:3n-6 + C22:4n-6. E Total n-3 PUFA: sum of C18:3n-3 + C18:4n-3 + C20:2n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3. a,b Means in the same row without same superscript differ significantly (P < 0.05). www.nature.com/scientificreports/ and leg muscles from chickens fed with these legumes than SBM; they also observed a reduction in the n-6/n-3 ratio. Recently, Paszkiewicz et al. 22 also found that the substitution of SBM by chickpea seeds can affect the FA proportions in the subcutaneous fat tissue of chickens. Desirable changes in the lipid profile of meat, including an increase in total n-3 PUFA content, and a decrease in the n-6/n-3 PUFA ratio, were also observed when SBM was replaced with camelina meal 23 or oil 24 and camelina oil or seeds 25 in diets for broiler chickens. On the other hand, Sirri et al. 26 reported that partial replacement of SBM with faba beans affected the proportions of some FA but had no effect on lipid fractions that are believed to be essential for human health. The reasons for the above contradiction could be different combinations of feed ingredients or level of inclusion used for formulating diets in each experiment, or even various genotypes of chickens. Table 4. Fatty acids composition (% of total fatty acids) in thigh muscles from 6-week-old broiler chickens. SEM standard error of the mean. A Group SBM: feed based on soybean meal; group CWP: feed based on raw cowpea seeds (Vigna unguiculata [L.] Walp, Aura 26 cv.). B Total SFA: saturated fatty acids, sum of C8:0 + C10:0 + C12:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0 + C24:0. C Total MUFA: monounsaturated fatty acids, sum of C14:1 + C15:1 + C16:1 + C17:1 + C18: 1n-9 + C22:1n-9 + C24: 1n-9. D Total n-6 PUFA: sum of C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:2n-6 + C22:3n-6 + C22:4n-6. E Total n-3 PUFA: sum of C18:3n-3 + C18:4n-3 + C20:2n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3. a,b Means in the same row without same superscript differ significantly (P < 0.05). www.nature.com/scientificreports/ On the basis of the obtained results, it can be concluded that the use of CWP as an alternative to SBM in the diet of broiler chickens had a favorable effect on meat quality. It is important because where CWP can be grown locally, low-input farming systems would benefit from using this ingredient for broiler chicken's feed. Additionally, CWP can be a cheaper source of PUFA and an efficient method for modifying the FA profile of the meat in a way that is beneficial according to the dietetic recommendations for humans.

Methods
The Animal Ethics Committee approved the study protocol of the National Research and Development Institute for Animal Biology and Nutrition (INCDBNA-IBNA) Baloteşti, Romania, under the EU Directive 2010/63/EU and Romanian Law on Animal Protection. The slaughter of birds was carried out following the applicable rules on handling animals at the time of slaughter, including humane treatment. Also, the methods used in the meat quality tests were carried out by the current and commonly used methodology described in the "Material and methods" section. The study was conducted in compliance with the ARRIVE guidelines. We confirm that all methods on chickens' were carried out in accordance with relevant guidelines and regulations.
The cowpea seed Aura 26 variety used in this study is part of the collection of genotypes from the Research-Development Station for Plant Culture on Sands, Dăbuleni, Romania, and the National Commission approved registration for horticultural plants, Decision No. 92841/2012.

Birds and diets.
The study was conducted on 240 one-day-old healthy broiler chickens (Ross 308) divided into two groups [a control group (SBM) feed a diet with soybean meal as a source of protein, and CWP group (experimental) feed a diet with untreated cowpea seeds], with similar initial weights (46.5 ± 0.23 g) into 6 replications with 20 birds each. The nutritional composition of CWP of the Aura 26 variety is described in Table 5. The feeding program was divided into two feeding phases: starter (days 1-24) and finisher (days 25-42). Diets for each feeding phase were formulated to be isocaloric, isonitrogenous, with similar total lysine, total sulphur amino acids (TSAA; Table 6), calcium and available phosphorous, and to meet or exceed breeder guidelines (Ross 308, Aviagen Ltd., Midlothian, UK). Diets were manufactured in mash form, without the inclusion of growth promoters or antibiotics. However, narasin as a coccidiostat (Monteban G100, Elanco GmbH) and phytase (Axtra PHY 5000 L, Danisco Animal Nutrition, Marlborough, UK) as exogenous enzymes were included in premixes of all two experimental diets. Feed and water were provided ad libitum.
Chickens were kept in pens on shavings litter in a temperature-controlled room with pens of identical size (1.75 × 1.55 m). Room temperature was maintained at 34 °C for the first 5 days and then gradually reduced according to standard management practices until a temperature of 22 °C by using thermostatically controlled heaters, fans, and adjustable sidewall inlets. Lighting was provided for 23 h/day from 1 to 7D, and from 8D, the  (WPSA, 1989). b Total n-6 PUFA, a sum of C18:2n-6; C20:2n-6. c Total n-3 PUFA, sum of C18:3n-3; C22:6n-3. Slaughtering procedures and muscle sampling. At 42 days of age, twelve birds (two birds/pen) from each feeding group (chosen based on pen average final live weight) were individually identified and weighed. The birds were electrically stunned before slaughter, exsanguinated by neck cut, scalded, and eviscerated. Chickens live weight was registered before slaughtering. A total of twelve breasts and twelve thighs were collected on their right and left sides, individually vacuum-sealed and refrigerated (4 ± 1 °C). Meat quality parameters (pH 24 , and color) were assessed on the Pectoralis major muscle (PM) on the right breast and the Biceps femoris muscle (BF) on the right thigh, while the left breast and thigh meat were frozen at − 20 °C until further analysis (instrumental TPA, proximate chemical composition and FA composition).

Meat quality traits. pH value and instrumental colour measurements.
After 24 h cold storage at 4 ± 1 °C, the pH value was measured in triplicate using a Hanna portable pH-meter (model HI 99163, Hanna Instruments, Romania), fitted with a spear-type electrode (FC 099 stainless steel blade tip) and an automatic temperature compensation probe. The color of the muscles was determined using a portable colorimeter (model CR 410, Konica Minolta Inc., Osaka, Japan) calibrated with a white ceramic tile on D65 illuminate. The results were expressed in the CIE Lab color space 27 . The lightness component is represented by L*, which ranges between 0 to 100, redness by a* and yellowness by b*, both of which have a range of −120 to + 120. For instrumental color determination, three measurements were performed on a fat-free surface area in different locations of each muscle (PM, BF).
Texture profile analysis. The texture measurements of raw chicken muscle samples were analyzed individually by a double cycle compression using a texture analyzer (Model CT3 BROOKFIELD Engineering Laboratories, Inc. MA, USA). To texture analysis, each muscle (PM and BF) was cut into 3-cylinder shapes with a diameter of 20 mm and a height of 15 mm. For an increased accuracy of the parameters reading, there were avoided any large areas of fat. The texture analyzer was equipped with a 50 kg load cell, a cylinder probe of 76.2 × 10 mm to com- www.nature.com/scientificreports/ press the samples and a fixture base table. The probe moved towards the sample at a constant speed of 2.0 mm s −1 (pre-test), 1.0 mm s −1 (test), and 2.0 mm s −1 (post-test). The data was collected using Texture Pro CT Software.
Proximate chemical composition. PM and BF samples were minced, homogenized, and divided into two parts. A portion was used to perform NIR (near-infrared reflectance) spectroscopy analysis. The remaining part was frozen and afterwards analyzed for total lipid extracts and FA composition. NIR data were acquired using a DA6200 meat analyzer (PerkinElmer, Inc. MA, USA), with transmission spectroscopy that uses diode array detectors in the wavelength range of 850 to 1050 nm. Raw minced and homogenized samples from the PM and BF muscles were loaded into a magnetically coupled plastic sample dish of 14 mm height and a volume of 170 mL and analyzed for moisture, protein, fat and collagen contents. To minimize sampling error, we set two duplicates, and each replication was measured twice. Before the sample measurements, a polystyrene check sample was used to verify the optical performance. The averaged spectrum was then used in subsequent analysis.
Fatty acid composition. The FA content was assessed via fatty acid methyl ester (FAME) gas chromatography. The method used for FA composition were done in the same way as previously described by Ciurescu et al. 28 .
In brief, FA from the total lipid extracts was converted to methyl esters by transesterification (in methanol containing 3% concentrated H 2 SO 4 , for 4 h at 80 °C). Methyl esters of FA were evaluated in a Perkin Elmer-Clarus 500 chromatograph equipped with a flame ionization detector (FID) fitted with a BPX70 capillary column (60 m × 0.25 mm × 0.25 µm film thickness). The column temperature was programmed at 5 °C/min from 180 to 220 °C. The carrier gas was hydrogen (35 cm/s linear velocities at 180 °C), and the splitting ratio was 1:100. The injector and detector temperatures used were 250 °C and 260 °C, respectively. Peaks were identified by injecting pure FAME standards; quantification was assessed using tridecanoic acid (C13:0) as an internal standard. The results were expressed as the percentage of the total detected FA. The ratio of n-6 PUFA to n-3 PUFA (n-6/n-3 PUFA ratio) was calculated.